Total parenteral nutrition (TPN) is designed to meet the nutritional requirements for humans and animals unable to obtain proper enteral nutrition orally or via the gastrointestinal tract. TPN solutions must provide all nutrients including carbohydrates, amino acids (as a substitute for protein), lipids, vitamins, and other essential compounds such as electrolytes and trace elements. The optimal desirable composition for TPN solutions is well known yet cannot always be achieved for each component because of intrinsic limitations imposed by the physiochemical properties of that component. Such limitations include poor solubility and instability during storage. In the case of TPN amino acid solutions, the optimal composition is one that produces a normal pattern of plasma amino acids (i.e., a normal plasma aminogram). The plasma amino acid levels are determined by the balance between the rate of administration of each amino acid and its rate of utilization. For example, a normal plasma aminogram corresponds to one produced after digestion of dietary protein and hepatic release of amino acids or one produced in normal breast-fed infants. Examples of normal plasma amino acid patterns in normal breast-fed infants is described by Wu, P. Y. K. (1986) J. Pediatr. 109: 347-349, and in adults is described by Perry, R. T. et al. (1969) Clin. Chim. Acta 25: 53-58.
However, because of the limited solubility of tyrosine and cyst(e)ine as well as the instability of cysteine asparagine and glutamine, solutions using free amino acids cannot be produced containing adequate, let alone optimal, amounts of these amino acids, as deduced from current knowledge of amino acid metabolism. Moreover, high levels of glutamate may lead to excitotoxicity, [Barinaga, M. (1990) Science 247: 20-22].
The relative insolubility of tyrosine in aqueous solutions at physiological pH has long presented problems in formulating TPN amino acid solutions. The ability to provide optimal tyrosine levels in TPN solutions is important in normalizing plasma levels of this amino acid. In infants, especially low-birth weight and premature infants, the metabolic pathway for conversion of phenylalanine, an essential amino acid, to tyrosine is not developed sufficiently to allow adequate conversion. Good tyrosine nutrition in early development may be crucial since it is a precursor of several hormones and neurotransmitters. Since the enzyme system which converts phenylalanine to tyrosine is primarily a liver enzyme, there may be particular disease conditions in adults, children and animals, especially liver diseases, in which the formation of tyrosine is impaired. Thus, the need for a TPN solution that achieves optimal (or adequate) plasma levels of tyrosine is highly desirable.
Typical amino acid solutions for TPN in pediatric patients contain tyrosine at about 44 mg/dl (e.g., Aminosyn-PF 10%, Abbott Laboratories), about the maximum amount soluble at the pH required for parenteral administration and an amount inadequate to attain normal plasma levels of tyrosine in patients, especially infants receiving TPN. Numerous alternatives have long been sought to increase tyrosine solubility or to provide other sources of tyrosine but none has satisfactorily solved the problem. The prior art teaches several soluble alternatives for tyrosine which can be formulated into TPN solutions, including use of high levels of phenylalanine, use of N-acetyl-L-tyrosine (NAcTyr), L-glycyl-L-tyrosine (GlyTyr), L-alanyl-L-tyrosine (AlaTyr) or general dipeptides containing tyrosine where the two amino acids have a normal peptide linkage joining the .alpha.-carboxyl group of the first residue and the .alpha.-amino group of the second residue and have the general formula X-Tyr or Tyr-Y wherein X is alanine, arginine, histidine, lysine, serine, glycine or glutamate and Y is arginine, histidine, glycine or glutamate. Of these dipeptides, all exhibit better aqueous solubility than tyrosine, and all suffer from instability in aqueous solution due to a tendency to form cyclic diketopiperazines. Of the known tyrosine-containing dipeptides, only AlaTyr was investigated for utility in TPN [Stegink, L. D. (1986) in Energy and Proteins Needs during Infancy, (S. J. Fomon and W. C. Heird, Eds.) Academic Press, Inc., NY, p183-206].
Formation of diketopiperazines may be a concern as illustrated in the case of aspartame, an unstable methyl ester of a dipeptide of aspartic acid and phenylalanine which limits the shelf-life of soft drinks in which it is used as a sweetener, because of loss of sweetness with formation of a diketopiperazine. While not a concern in foods ingested orally, data establishing the safety of diketopiperazines administered intravenously, as in TPN into very small infants, is unavailable.
Aminosyn-PF 10% contains high levels of phenylalanine based on the assumption that phenylalanine can serve as a precursor for tyrosine. While this may be a fair assumption for some adults, newborn infants appear unable to convert significant amount of phenylalanine into tyrosine. For example, breast-fed infants have a plasma ratio of phenylalanine to tyrosine (Phe/Tyr) of about 0.6, low birthweight infants fed pooled human milk have a ratio of about 0.7-0.8, and infants fed solely by TPN, using amino acid mixtures like Aminosyn-PF 10% or other compositions presently available, have plasma Phe/Tyr ratios that are abnormally high, ranging from about 2.2-3.7. Since phenylalanine and tyrosine compete for transport from the blood into tissues, including the brain, these high levels of phenylalanine relative to tyrosine only exacerbate the deficit in tissue tyrosine. This can clearly compromise the growth and development of the infant.
Moreover, there are also disease conditions in adults and children, such as those involving impairment of liver function, where metabolic conversion of phenylalanine to tyrosine may be disturbed. Such patients would benefit from improved TPN solutions supplying adequate amounts of tyrosine. Hence, replacement of tyrosine by phenylalanine may be counterproductive as a method to increase plasma tyrosine levels.
Another source of tyrosine examined because of its increased aqueous solubility, and which avoids the problem of diketopiperazine formation, is NAcTyr. The use of NAcTyr in TPN for pre-term neonates has been reported (Helms, R. A. et al. (1987) J. Pediatr. 110: 466-470). A study of NAcTyr utilization in TPN by Magnusson, I. et al. (1989) Metabolism 38: 957-961, showed that in adults the plasma levels of tyrosine four hours after administration of 5 g tyrosine in a 10 mg/ml solution were nearly the same as the basal tyrosine levels (63 vs. 51 .mu.mol/l, respectively). However, while the NAcTyr levels increased dramatically in the same time frame (from 9 to 256 .mu.mol/l), 56% of the administered NAcTyr was excreted in the urine within 4 h. In another study by Stegink, supra, rats infused with NAcTyr at a rate of 0.5 mmol/kg/day or 2 mmol/kg/day showed that after 24 h of TPN, the plasma tyrosine levels were unchanged at the low infusion rate and merely increased two-fold at the higher rate. Although the studies using NAcTyr in rats indicate some utilization of the tyrosine, there appears to be a species difference between the rat and the human, since humans cannot release tyrosine efficiently from NAcTyr. Thus, despite its increased solubility, NAcTyr is not satisfactory to replace or supplement tyrosine in TPN solutions. NAcTyr suffers the further disadvantage of not being a normal product of human metabolism, and therefore the safety of its long term use, especially in high risk infants, is a concern.
AlaTyr has also been investigated as an alternative source of tyrosine in amino acid solutions for TPN (Stegink, supra). Like NAcTyr, AlaTyr is sufficiently soluble under aqueous, physiological conditions to deliver potentially adequate nutritional levels of free tyrosine. However, administration of AlaTyr to rats at a rate of 0.5 mmol/kg/day or 2 mmol/kg/day indicated that after 24 h of administration, the plasma tyrosine levels were unchanged at the lower rate and merely increased two-fold at the higher rate. Renal excretion of AlaTyr also occurred but at a slightly lower rate than NAcTyr loss. AlaTyr as well as the soluble dipeptides discussed above suffer a major disadvantage in that they are unstable in aqueous solution, especially upon the prolonged storage periods to which TPN amino acid solutions are often subjected. This instability appears to be caused by diketopiperazine formation (Stegink, supra). Hence, .alpha.-carboxyl-linked peptides cannot be added to TPN amino acid solutions subjected to long storage periods and are, thus, best added just prior to administration of the TPN solution, a practice that leaves room for error and contamination.
In a survey of di- and tri-peptides for TPN, a large number of glycyl-Z dipeptides were examined for utility in TPN [Adibi, S. (1987) Metabolism 36:1001-1011], where Z was one of the 20 common amino acids. In particular, upon administration of AlaTyr or GlyTyr in rats at a rate of 0.5 mmol/kg, plasma tyrosine levels did not increase as rapidly for GlyTyr as for AlaTyr. In both cases, the levels reached the same value at longer times. As mentioned above, the GlyTyr dipeptide also suffers the disadvantage of being unstable during storage in aqueous solution.
Accordingly, the present invention provides a soluble source of tyrosine which does not exhibit the disadvantages of the compounds known in the prior art for TPN. The subject tyrosine source, .gamma.-GluTyr, readily supplies adequate and optimal amounts of tyrosine to the patient, is stable upon prolonged storage periods in aqueous solutions used for TPN since it does not contain an .alpha.-carboxyl linkage, and is a naturally occurring dipeptide, being generated during the .gamma.-glutamyl cycle as described by Meister (1973) Science 180 33-39. .gamma.-GluTyr is readily metabolized to release free tyrosine at least in part via degradation by .alpha.-glutamyl transpeptidase. Since .gamma.-GluTyr is a normal product of metabolism, it provides a safe source of tyrosine in vivo, with little potential for producing toxicity in high-risk infants and other patients, including humans and animals.
Like tyrosine, cysteine has been difficult to supply in adequate amounts via TPN. When supplied as cysteine in an aqueous solution at neutral pH in the presence of oxygen, cysteine is spontaneously converted to cystine with release of hydrogen peroxide as shown below: ##STR1## The designation cyst(e)ine refers either to the oxidized or reduced form of cysteine. Cystine is quite insoluble in water (1 mg/dl) especially at the neutral pH required for TPN. Thus, despite the solubility of cysteine, its conversion to cystine coupled with the insolubility of cystine, makes it difficult to supply adequate cysteine by TPN.
Although cyst(e)ine is not considered a dietary "essential" amino acid for children or adults, it may be essential for neonates. This amino acid is formed via a metabolic pathway called "trans-sulfuration." In this process the "essential" amino acid, methionine, donates its sulfur atom to serine, forming cysteine. The metabolic pathway to cysteine, which involves five different enzyme-catalyzed reactions, is shown below in abbreviated form: ##STR2## Cystathionase, the enzyme which catalyzes the final step in the biosynthesis of cysteine, is primarily a liver enzyme and is fully operative only after birth. Thus, the neonate, and particularly the pre-term neonate, cannot meet the need for cysteine via the normal biosynthetic route. The intermediate cystathionine accumulates and is excreted in the urine, thus causing cysteine to become a nutritionally "essential" amino acid for these infants.
Cysteine has a number of important intracellular functions in addition to its role in protein synthesis: (a) Cysteine is required for the conversion of the vitamin, pantothenic acid, to coenzyme A, its metabolically active form. (b) Cysteine is a metabolic precursor of the amino sulfonic acid, taurine. Taurine is currently included in TPN solutions, reducing some of the dietary need for cysteine. (c) Cysteine is limiting for the biosynthesis of the tripeptide, glutathione (gamma-glutamyl-cysteinylglycine), which plays a major role in protecting tissues against oxidative damage. Glutathione (GSH) is also important in the detoxification of xenobiotics and in the maintenance of functional thiol groups in proteins. [Meister, A. et al. (1983) Ann. Rev. Biochem. 52: 711-760].
Water-soluble GSH, and fat-soluble vitamin E, are important antioxidants and may be of special significance in protecting infants exposed to hyperbaric oxygen. A cysteine deficiency can lead to export of GSH from the liver to replenish plasma cyst(e)ine through degradation of plasma GSH [Meister, A. (1988) J. Biol. Chem. 263: 17205-17208]. Depletion of liver GSH below a critical level may lead to numerous matabolic aberrations.
One major concern in the delivery of cyst(e)ine via TPN is that this amino acid has been shown to be lethal when fed to weanling rats at a level of 15.7 g N/kg basal diet, and neurotoxic when administered in a single subcutaneous dose (1.2 mg/kg body weight) to 4-day-old rats, and in a single intraperitoneal dose (10 mmol/kg body weight) to mice [Anderson, M. E. et al. (1987) Methods Enzymol. 143: 313-325]. The reasons for this toxicity are not clear, but it appears to be associated with extracellular cyst(e)ine. Thus, a means of delivering cyst(e)ine intracellularly is desired.
Several methods have been used or suggested in the prior art for provision of adequate cysteine during TPN. However, these methods suffer many disadvantages which can be overcome by providing .gamma.-GluCys for use in TPN solutions.
Cysteine-hydrochloride (cysteine-HCl) has been administered as a separate solution, not combined in the mixture of the other amino acids used in TPN. This soluble form of cysteine is stable at low pH. The amount of HCl which high-risk infants can tolerate is limited and this, in turn, limits the amount of cysteine-HCl which may be used in TPN. Cysteine-HCl in TPN has been implicated in the production of acidosis in some treated low-birth-weight infants [Heird, W. C. (1988) Pediatr. 81: 41-50].
Another source of cysteine examined for use in TPN has been N-acetylcysteine (NAcCys). However, like NAcTyr, NAcCys was not found to be a satisfactory replacement source for cysteine (Magnussen et al.). In particular, the plasma levels of cysteine four hours after administration of 5 g cysteine in a 200 mg/ml solution decreased relative to the basal cysteine level (134 vs 207 .mu.mol/l). However, while the NAcCys levels increased dramatically in the same time frame (from 2 to 488 .mu.mol/l), 11% of the administered NAcCys was excreted in the urine within 4 h. Stegink et al. also reported large urinary losses of N,N'-bis-acetylcystine when administered for TPN and concluded that this compound was not a suitable alternative source for cysteine in TPN.
Further to the Adibi et al. study of di- and tri-peptides in TPN as described above, no dipeptides containing cysteine having utility in TPN were disclosed.
GSH has also been used as a source of cysteine during long-term TPN in the growing rat [Neuhauser-Berthold, M. et al. (1988) Metabolism 37: 796-801]. There have been no reports of GSH stability upon prolonged storage under TPN storage conditions. Further, GSH does not appear to be transported into cells whereas .gamma.-GluCys derivatives are transported (as .gamma.-L-glutamyl-L-cystine, i.e., .gamma.-Glu(Cys).sub.2 ; or N,N'-bis-(.gamma.-L-glutamyl)cysteine, i.e. (.gamma.-GluCys).sub.2) [Anderson, M. E. et al. (1983) Proc. Natl. Acad. Sci. USA 80: 707-711. Thus .gamma.-GluCys and its derivatives may provide a more efficient means to increase the GSH content in tissues as well as to provide a stable source of cysteine.
A further concern in current TPN formulations is the inclusion of high levels of methionine in these solutions, with the misguided view that large supplements of methionine will substitute for the inadequate cysteine levels in TPN solutions. High intake of methionine is associated with hepatotoxicity [Benevenga, N.J. (1974) J. Agric. Food Chem. 22: 2-9]. In view of this, there is a alarming discrepancy between reported plasma ratios of cysteine to methionine (Cys/Met) of 10/1 in breast-fed infants [Gaull, G. E. et al. (1977) J. Pediatr. 90: 348-355] and of 0.6 in infants on TPN supplemented with L-cysteine-HCL [Zlotkin, S. H. et al. (1981) Am. J. Clin. Nutr. 34: 914-923]. The use of .gamma.-GluCys and derivatives in TPN solutions make it possible to increase the cysteine supply in a non-toxic form, and to reduce the amount of methionine needed in these solutions to achieve more normal Cys/Met ratios.
Accordingly, the present invention provides a soluble source of cysteine which does not exhibit the disadvantages of the compounds known in the prior art for TPN. The subject cysteine source, .gamma.-GlyCys and derivatives described below, readily supplies adequate and optimal amounts of cysteine to the patient, is stable upon prolonged storage periods in aqueous solutions used for TPN since it lacks an .alpha.-carboxyl linkage. Moreover, like .gamma.-GluTyr, .gamma.-GluCys is a naturally occurring dipeptide, which can be generated by the tissue enzymes, .gamma.-glutamyl transpeptidease or by .gamma.-glutamylcysteine synthetase. As a normal product of metabolism, .gamma.-GluCys provides a safe source of vivo, with little potential for producing toxicity in high risk infants and other patients, including humans and animals.
Glutamine is yet another amino acid which has been difficult to supply in adequate amounts via TPN. Although glutamine is present in plasma at the highest concentration of any amino acid, glutamine is not included in TPN because of its instability in aqueous solutions. In particular, glutamine breaks down in aqueous solution to form pyroglutamic acid with a release of toxic ammonia according to the reaction below: ##STR3## Hence, TPN solutions containing glutamine which are stored even for short lengths of time can accumulate toxic ammonia. While a fresh glutamine solution can be added to the TPN solution, this greatly increases the risk of contamination and error in formulation. Thus, TPN solutions in present use do not contain glutamine.
Because glutamine cannot be included in mixtures of amino acids for TPN, high levels of glutamate are substituted on the assumption that in vivo conversion of glutamate to glutamine occurs. However as discussed below high levels of glutamate are neurotoxic and should be avoided. The normal plasma ratio of glutamine (Gln) to glutamate (Glu), based on mean values is about 27:1 (Perry et al. (1969) Clin. Chim. Acta 25:53-58), whereas in infants maintained for one week on TPN, the Gln:Glu ratio is reduced to 1.1:1 (Aminosyn PF) and 0.7:1 (Neopham) (Coran et al. (1989) J. Pediatr. Enter. Nutr. 11:368-377). This reduction appears to be due to both a decrease in plasma glutamine and an increase in plasma glutamate.
The markedly reduced ratio of plasma Gln:Glu does not provide sufficient glutamine for proper nutrition of the gut. Lack of glutamine appears to be a factor in gut pathology associated with the difficulty many infants experience in adapting to oral feeding after prolonged TPN. In fact, studies in rats showed that TPN lacking glutamine lead to decreased villus height in the intestine, whereas inclusion of glutamine in TPN preserved the normal architecture of gut villi (Surg. Form. 37:56-58 (1986)). In these studies freshly prepared glutamine was added to the TPN mixture.
One method used in the prior art to supply glutamine has been via the dipeptides glycylglutamine (GlyGln) and alanylglutamine (AlaGln) (Adibi, supra). Like other dipeptides these compounds are also unstable during prolonged storage in aqueous solution due to the tendency to form cyclic diketopiperazines.
Accordingly, the present invention provides a stable source of glutamine which does not exhibit the disadvantages of the compounds known in the prior art for TPN. The subject glutamine source, .gamma.-GluGln, readily supplies adequate and optimal amounts of glutamine to the patient, is stable upon prolonged storage periods in aqueous solutions used for TPN since it does not contain an .alpha.-carboxyl linkage, and is a naturally occurring dipeptide, being generated during the .gamma.-glutamyl cycle as described by Meister, supra. .gamma.-GluGln is readily metabolized to release free glutamine, at least in part via degradation by .gamma.-glutamyl transpeptidase. Since .gamma.-GluGln is a normal product of metabolism, it provides a safe source of glutamine in vivo, with little potential for producing toxicity in high-risk infants and other patients, including humans and animals.
Another important advantage in the use of .gamma.-GluTyr .gamma.-GluCys and .gamma.-GluGln in TPN is that upon hydrolysis in vivo, glutamic acid is gradually released. This allows reduction of the rather large amount of free glutamic acid normally present in TPN solutions (for example there is 820 mg/dL in Aminosyn-PF 10%). Thus, glutamic acid can be reduced proportionately by the amount administered as .gamma.-GluTyr, .gamma.-GluCys or .gamma.-GluGln. Reduction of free glutamic acid in TPN is important in light of the concern about the excitotoxicity and neurotoxicity of free glutamic acid especially as related to the use of monosodium glutamate (MSG) as a food additive. The safe use of glutamic acid, which has been called an "excitotoxin," should be considered in determining the amounts of glutamic acid administered by TPN to infants, who may be more susceptible than adults to nerve damage by glutamate (Barinaga supra). Thus, in addition to the benefits relative to stability and solubility of tyrosine, cysteine and glutamine, the present invention provides a means to reduce free glutamic acid in TPN solutions while still providing adequate nutritional levels of glutamic acid.